1. Field of the Invention
The present invention relates generally to compositions for modulating the level of translation of a polypeptide, and more specifically to synthetic internal ribosome entry site (IRES) nucleotide sequence and to methods of identifying a synthetic IRES.
2. Background Information
Molecular biology provides tools for genetically engineering organisms to express large amounts of useful gene products, including new and useful gene products. As such, medically useful gene products such as growth factors, hormones, and immunoregulatory polypeptides, for example, interferons and interleukins are now available in sufficient quantities for therapeutic use. In addition, molecular biology has allowed gene therapy to evolve to a form that it promises to provide new ways for doctors to manage previously untreatable conditions.
Although molecular biology currently provides tools for expressing recombinant proteins in the cells of bacteria, plants, and animals, including human cells, methods for manipulating the amount of a protein that is expressed in the cells are limited. For example, the recombinant protein can be expressed from a particular gene promoter, that has a known level of activity or that is expressed only in one or a few cell types. However, while, the use of a particular promoter may result in the expression of a particular amount of RNA encoding the protein, the amount of protein translated from the RNA does not always correlate directly with the amount of RNA that is expressed. For example, some of the RNA may be degraded in the particular cell, or the RNA may not be translated efficiently. In other cases, the protein may be translated in an amount that is toxic, either to the cell expressing the protein or to another cell that is in proximity to a cell that produces and secretes the protein.
Nucleotide sequences that are involved in regulating the translation of a polypeptide have been described, and can be utilized to help regulate the amount of a protein translated from an RNA molecule. Some of these nucleotide sequences are functional only when positioned at the 5′ end of an RNA molecule, and are useful for regulating the expression of a protein encoded by the nucleotide immediately downstream of the translation regulatory element. However, such nucleotide sequences are not useful for regulating the expression of a number of proteins that are encoded by a single RNA molecule containing a series of open reading frames. For this purpose, nucleotide sequences that can regulate translation from within a nucleotide sequence can be useful. However, only a few such sequences, referred to as internal ribosome binding sites (IRES), have been described, and they generally are very large, containing more than a hundred, generally several hundred, nucleotides.
The large size of the known IRES sequences limits their usefulness. For example, many gene therapy vectors such as retrovirus vectors are limited in the size of a polynucleotide insert that can be contained in and expressed from the vector. As such, polynucleotides that encode very large proteins cannot be used with certain vectors. The use of an IRES, which can consist of several hundred nucleotides, only further limits the size of the encoding polynucleotide that can be included in the vector, thus further limiting the polynucleotides that can be used in a gene therapy procedure. Furthermore, the use of a single IRES may not result in sufficient expression of a desired polypeptide. Thus, a need exists for regulatory elements that are useful for modulating the level of protein expression. The present invention satisfies this need and provides additional advantages.